Under certain conditions, adsorbent materials containing ionic groups can bind molecules of a net opposing charge. Such processes, for example chromatography, are currently utilised in the purification and separation of biomolecules in a complex mixture such as blood or fermentation or cell culture broths.
In column chromatography, the mixture to be analysed is applied to the top of a column comprising an adsorbent material which acts as the “stationary phase”. A liquid solvent (the “mobile phase”) is passed through the column under gravity or pressure carrying the dissolved mixture. Because the different compounds in the mixture have different ionic interactions with the mobile and stationary phases, they will be carried along in the mobile phase to varying degrees, resulting in separation. A salt gradient is then usually applied to remove, in turn, separate bound components.
In such processes, the exact conditions for separation are typically determined by trial and error. The operational selectivity of an adsorbent relates to the number of molecules that bind to it as the mixture passes over; under normal conditions there is generally low specificity. Variation in the pH or ionic strength causes the interaction between individual components of the mixture and adsorbent to change. The ionic strength may be varied to allow a desired component to adsorb, but so that solvent molecules or additional components compete for available binding sites on the adsorbent, thus preventing the binding of an undesired component. Selectivity also varies depending on the physical structure of the adsorbent, for example the size distribution of pores or the chemical nature of the underivatised adsorbent.
The “working capacity” (dynamic capacity) of an adsorbent refers to the amount of a particular component which will bind to and be retained on the adsorbent. Working capacity is dependent on, inter alia, the charge density, ligand type and pore size distribution of the adsorbent.
Chang et al [Journal of Chromatography A, 827 (1998), 281-293] suggests that the protein adsorption capacity of an adsorbent is strongly correlated with the accessible surface area, and less so with the intrinsic adsorption affinity. The authors propose that uptake dynamics are influenced to a large extent by mean pore size, although it is acknowledged that other structural parameters, such as pore connectivity and adsorption affinity, may also play a role.
DePhillips et al [Journal of Chromatography A, 933 (2001), 57-72] reports that, above a threshold amount, increased charge density and ionic capacity do not necessarily result in increased protein retention. DePhillips et al postulate that a high charge density is relatively unimportant, proposing that an equivalent capacity may be attained by optimally orientating and positioning ligands of lower charge density. The data presented are based on experiments using additional salt to optimise the ionic strength.
In summary, research in this field has, over time, suggested that an increase in the charge density of the adsorbent leads, up to a point, to an, increased capacity, when added competing salts are used.
Analytical and production-scale chromatography differ, inter alia, in the way in which their performance is measured. For analytical separations, the overriding requirement is that analysis is rapid; this is often achieved using small, non-porous stationary phase particles. For large-scale chromatographic processes, capacity, recovery and throughput are typically the factors on which performance is judged. Optimum performance may be achieved by trading off throughput against selectivity and recovery. Currently, desirable working capacity is attained by using adsorbents of high charge density, with additional salt or buffer incorporated in the mobile phase. As mentioned above, the presence of additional salt (e.g. NaCl) reduces the strength of interaction between the stationary and mobile phase, allowing the specific binding of a particular component.
Current methodologies suffer from a number of limitations, one of which is cost. A major factor in the manufacture of biomolecules is the cost of raw materials. Buffered solutions are required to stabilise biomolecules against variations in pH and to reduce the likelihood of insolubilisation (precipitation). For this reason, concentrations of buffer/salt components are kept to a minimum in feed solutions and usually range from 10 to 100 mM, depending on the biomolecule to be stabilised.